Evolution of gene regulatory networks controlling post-embryonic morphogenesis Morphogenesis, or the development of form, is a universal process during development of multicellular organisms that is controlled by the precise spatiotemporal expression of genes within gene regulatory networks (GRNs). While advances have been made in elucidating GRNs that control embryonic development in model organisms, we lack an understanding of how GRNs regulate post-embryonic morphogenesis and how these networks evolve. The hotspot hypothesis predicts that the architecture of GRNs can bias evolution, such that morphologies evolve via repeated co-option of a master regulator (i.e. a gene that is required and sufficient for morphogenesis). This proposal will use a post-embryonic morphogenic process, known as Tail Tip Morphogenesis (TTM), which evolved multiple times independently in Caenorhabditis elegans and related species, to investigate the architecture and evolution of GRNs, and test the hotspot hypothesis. In C. elegans, DMD-3, a DM-domain transcription factor, is the master regulator within the GRN governing TTM. Aim 1 uses single-tissue RNA-Seq in a time series over the course of TTM in lineages where TTM independently evolved. Then computationally infer the GRN underlying TTM in each species. The inferred GRNs will be used to test the hotspot hypothesis and will contribute to our general understanding of how GRNs drive morphogenesis and how plastic GRN architectures can be. Aim 2.1 validates the hotspot hypothesis by knocking out dmd-3, or another candidate regulator inferred from Aim 1, in species where TTM independently evolved. Aim 2.2 validates the predicted downstream interactions within the GRN by using single-tissue RNA-Seq on the regulator knockout lines. Aim 3 investigates the functional role of conserved modules (i.e. sets of genes and their interactions) within the GRNs that have human homologs. As DMD-3 is a homolog to DMRT-1, required for male fates in humans, regulators and effectors of DMD-3 could be candidate targets for future drugs or therapies that could, for example, help people with sex reversal. Additionally, because morphogenesis is a universal developmental process, this work will also likely identify genes that are conserved in other morphogenic processes, such as cancer metastasis, regeneration, and wound healing. This project will be conducted within the Center for Developmental Genetics at New York University, a world- renowned research institution with top-notch resources and faculty, under the advisement of Prof. David Fitch who has 25 years of experience as a researcher, mentor, and educator in the field of evolutionary developmental biology. My training goals are to 1) expand my knowledge in developmental biology, 2) learn developmental genetics wet lab techniques, 3) continue my education in bioinformatics, 4) develop teaching and mentoring skills, 5) develop species related to C. elegans into satellite model systems to use in my independent research career. These goals will be achieved using the above research as a platform in addition to mentoring from Prof. Fitch, workshops, conferences, and mentoring undergraduates.